3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized by the sequential action of the two "sulfate activating enzymes": ATP sulfurylase and APS kinase. PAPS serves as the sulfate donor for the biosynthesis of all known sulfate esters. It is involved in the formation of the essential acid proteoglycans (e.g. chondroitin sulfate and dermatan sulfate) and the structurally- related anticoagulant, heparin PAPS is also involved in the biosynthesis of phenol sulfates, ascorbic acid-2-sulfate, and Vitamin D sulfate. Sulfation may play a role in regulating the active levels of epinephrine, tyramine, serotonin and enkephalin. Recently, it has been shown that sulfation facilitates the biological deiodination of thyroxine. Sulfated glycoproteins are produced by many animal cells and may be involved in tissue differentiation. In spite of the importance of the sulfate activating enzymes, there have been no detailed studies on homogeneous preparations of ATP sulfurylase from any animal source. Similarly, nothing about the molecular properties of pure APS kinase has been published. Therefore, we propose to examine ATP sulfurylase and APS kinase from rat and (if feasible) human liver. The physical, chemical, structural, kinetic, and regulatory properties of the enzymes will be determined. We will also investigate the possible hormonal control of the sulfate activating enzymes. One of the important functions of sulfation in vivo is to detoxify the endogeneously formed hepatotoxic monohydroxy bile salts. Monohydroxy bile salts have been shown to produce lesions of the liver and biliary tract, and intrahepatic cholestasis. Addition of a sulfate group to these compounds increased their polarity and enhances renal and fecal excretion. Bile salt sulfotransferase (BSS), the enzyme catalyzing the transfer of a sulfate group from PAPS to bile salts, has been found in liver cytosol of various mammals including man. Recently we have demonstrated the existence of two forms of BSS in rat liver. In this proposed studies, we will continue to characterize and compare these isozymes from rat with respect to their kinetic constants, subunit structure, and amino acid composition, and most importantly, the substrate preferences of each isozymes. We will also investigate the effect of experimental cholestasis on BSS activity and isozyme patterns. In addition, we will further characterize the physiochemical properties of BSS from human liver.